Quinazolines for treating brain tumor

ABSTRACT

Novel substituted quinozaline compounds and conjugates useful to inhibit the growth of brain tumor cells and to inhibit adhesion and migration of brain tumor cells. The compounds of the invention include 4-(3′-bromo-4′-hydroxy phenyl)-amino-6,7-dimethoxyquinazoline and this compound covalently bound to EGF.

This application in a continuation of application Ser. No. 09/361,088now U.S. Pat. No. 6,316,454, filed Jul. 26, 1999 which is a continuationof application Ser. No. 09/087,479 now abandoned, filed May 28, 1998which application are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel quinazoline derivatives effective toinduce apoptosis of brain tumor cells. In particular, the inventionincludes novel hydroxy quinazoline derivatives having potentcytotoxicity against human brain tumor cells, including glioblastoma.The novel compounds of the invention further inhibit adhesion of braintumor cells to extracellular matrix proteins and inhibit migration ofbrain tumor cells through the extracellular matrix, activities requiredfor tumor metastases.

BACKGROUND OF THE INVENTION

As the most malignant primary central nervous systems tumors, high gradeanaplastic astrocytoma and glioblastoma multiforme respond poorly tocontemporary multimodality treatment programs employing surgicalresection, radiation therapy and chemotherapy with a median survival ofless than one year after initial diagnosis (Pardos, et al., 1997, CancerMedicine, 1:1471-1514; Brandes, et al., 1996, Cancer Invest. 14:551-559;Finlay, J. L., 1992, Pediatric Neuro-Oncology, 278-297; Pardos, et al.,1998, Sem. Surgical Oncol., 14:88-95). Consequently, the development ofeffective new agents and novel treatment modalities against these verypoor prognosis brain tumors remains a major focal point in translationaloncology research.

Glioblastoma multiforme is also a highly invasive primary brain tumorwith a disappointingly high local recurrence rate and mortality. Newagents capable of inhibiting the infiltration of normal brain parenchymaby glioblastoma cells are urgently needed.

SUMMARY OF THE INVENTION

In a systematic effort to identify a cytotoxic agent with potentanti-tumor activity against glioblastoma cells, severalhydroxy-substituted quinazoline-derivatives were synthesized andexamined for their in vitro and in vivo effects on human glioblastomacells. Novel hydroxy- and halo-hydroxy-quinazoline derivatives werefound to exhibit potent cytotoxic activity against human glioblastomacells at micromolar concentrations. Targeting of these compounds to thesurface of brain tumor cells, for example, by conjugating hydroxy- andthe halo-hydroxy compounds to a targeting moiety such as epidermalgrowth factor (EGF), further enhanced the cytotoxic activity (atnanomolar concentrations) The conjugate demonstrated more rapid and morepotent anti-brain tumor activity, including apoptotic death ofglioblastoma cells in vitro, significantly improved tumor-free survivalin an in vivo SCID mouse glioblastoma xenograft model, inhibition oftumor cell adhesion to ECM proteins, and inhibition of tumor cellmigration and invasion activity.

Accordingly, the present invention includes novel compounds andcompositions having potent cytotoxic activity against brain tumor cells.Compositions of the invention contain an effective cytotoxic orinhibitory amount of a hydroxy-substituted quinozaline compound, moreparticularly of a hydroxy- or halo-hydroxy-substituted quinazolinederivative. The compounds of the invention include those having thefollowing formula:

where X is HN, R₁₁N, S, O, CH₂, or R₁₁CH, and one or more of R₁, R₂, R₃,R₄, R₅ is OH, SH, or NH₂. Preferred embodiments include those where X isHN; R₃ is OH; R₂ and/or R₄ is a halogen, preferably Br. In anotherpreferred embodiment, one or more of R₁-R₅ form a second ring fused tothe phenyl ring, for example, forming a napthyl ring and having at leastone hydroxy substitution.

Preferred cytotoxic compounds of the invention include4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P154],4-(4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P131], and4-(2′-Hydroxy-naphthyl-3′)-amino-6,7-dimethoxyquinazoline[WHI-P292].

The compounds of the invention can be formulated for delivery to asubject as a pharmaceutical composition, and can preferably be modifiedfor selective killing of brain tumor cells by conjugation to a cellspecific targeting moiety, such as an anti-cell surfaceantigen-antibody, or a moiety known to bind a cell surface receptor,such as EGF. The compounds of the invention are preferably covalentlybonded to the targeting moiety. One exemplary targeting moiety is EGF,which, when conjugated to the compound of the invention, rapidly andspecifically directs the compound to brain tumor cells expressing theEGF receptor, resulting in specific, rapid, and enhanced cytoxicity.

The compounds of the invention are administered to a subject to inhibitthe growth of brain tumor cells, to induce apoptosis of brain tumorcells, thereby reducing tumor mass. Compounds of the invention are alsoadministered to inhibit the adhesion and migration of brain tumor cells,for example, inhibiting the infiltration of normal brain parenchyma byglioblastoma cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are cell survival graphs demonstrating the cytotoxicactivity of WHI-P154 and other compounds of the invention against U373glioblastoma cells.

FIGS. 2A and 2B are photographs of laser analyzed immunostained cellsdemonstrating cell surface expression of EGF receptor on U373 and U87human glioblastoma cells.

FIGS. 3A, 3A′, 3B, 3B′, 3C, 3C′, 3D, and 3D′ are photographs ofimmunostained cells showing EGF-R mediated uptake of EGF-P154 by U373human glioblastoma cells.

FIGS. 4A and 4B are photographs showing uptake of WHI-P154 and EGF-P154by human glioblastoma cells.

FIG. 5A is a graph demonstrating the cytotoxic activity of EGF-P154against brain tumor cells. FIG. 5B shows the activity of unconjugatedWHI-P154 and EGF-P154 on EGF-R negative leukemia cells.

FIGS. 6A-6E are photographs showing morphological features of gliomacells treated with EGF and EGF-P154.

FIGS. 7A and 7B are graphs showing tumor volume and tumor-free survivaltimes in a SCID mice human glioblastoma model treated with WHI-P154 andEGF-P154.

FIG. 8 is a bar graph showing adhesion of glioblastoma andmedulloblastoma cells to plates coated with various ECM proteins.

FIGS. 9A-9D are bar graphs showing inhibition of cell adhesion to ECMproteins in the presence of the compounds of the invention.

FIG. 10 is a graph showing the inhibition of EGF-induced cell adhesionin the presence of the compounds of the invention.

FIGS. 11A and 11B are graphs demonstrating inhibition of brain tumorcell migration through the extracellular matrix MATRIGEL Matrix in thepresence of the compounds of the invention.

FIGS. 12A-C and 12A′-C′ are photographs of immunostained cellsdemonstrating the inhibition of focal adhesion complex formation inglioblastoma cells in the presence of the compounds of the invention.

FIGS. 13A-13C are photographs of immunostained cells demonstrating theinhibition of actin stress fiber formation in glioblastoma cellscontacted with the compounds of the invention.

FIGS. 14A-D are photographs showing inhibition of tumor cell outgrowthfrom glioblastoma spheroids in the presence of the compounds of theinvention.

FIG. 15 is a graph demonstrating the cytotoxic effect of WHI-P292 onglioblastoma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes novel hydroxy-substituted quinazolinederivatives having potent activity as cytotoxic agents against braintumor cells, including glioblastoma cells. In addition, thehydroxy-substituted quinazoline compounds of the invention are potentinhibitors of tumor cell adhesion and migration, activities required fortumor cell metastases.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “alkyl” includes both branched and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. As a preferred embodiment, chains of 1 to 4 carbon atomsare included, for example methyl, ethyl, propyl, isopropyl, butyl,secondary butyl, t-butyl, and the like.

As used herein, “alkene” includes both branched and straight-chainunsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms, preferably chains of 1 to 4 carbon atoms.

As used herein, “acyl” includes —C(O)R, where R is H, alkyl, or arylcontaining 1 to 4 carbon atoms.

As used herein “halogen” includes fluoro, chloro, bromo, and iodo. Apreferred halogen or halo substituent is Br.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with a compound of the invention, allowsthe compound to retain biological activity, such as the ability toinduce apoptosis of brain tumor cells, and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsions, andvarious types of wetting agents. Compositions comprising such carriersare formulated by well known conventional methods (see, for example,Remington's Pharmaceutical Sciences, Chapter 43, 14th Ed., MackPublishing Co., Easton, Pa.).

Compounds of the Invention

The novel substituted quinazolines of the invention have the generalstructure represented by the following formula I:

where X is selected from the group consisting of HN, R₁₁N, S, O, CH₂,and R₁₁—CH. R₁₁ is H, alkyl, having 1 to 4 carbon atoms, or acyl.Preferably, X is NH; and preferably R₁₁ is H.

R₁, R₂, R₃, R₄, R₅, R₆, R₇ and R₈ are each independently selected fromthe group consisting of H, OH, SH, NH₂, NO2, alkoxy, alkylthio andhalogen. R₉ and R₁₀ are each independently selected from the groupconsisting of H, alkyl or acyl, containing up to 4 carbon atoms.Preferably, R₉ and R₁₀ are methyl.

At least one of R₁, R₂, R₃, R₄, R₅ is OH. Alternatively, at least one ofR₁-R₅ is a compound such as SH or NH₂.

In an alternative embodiment, one or more of R₁-R₅ forms a second ringfused to the phenyl ring. For example, the following compounds includesecond rings fused to the phenyl ring via one or more of R₁-R₅:

Exemplary Compounds

Some exemplary compounds of the invention are listed below with theircharacterization data:

4-(3′,5′-Dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P971]

yield 72.80%; m.p.>300.0° C. UV(MeOH)λ_(max): 208.0, 210.0, 1245.0,320.0 nm; IR(KBr)υ_(max): 3504(br), 3419, 2868, 627, 1512, 1425, 1250,1155 cm⁻¹; ¹H NMR(DMSO-d₆): δ 9.71(s, 1H, —NH), 9.39(s, 1H, —OH),8.48(s, 1H, 2-H), 8.07(s, 2H, 2′,6′-H), 7.76(s, 1H, 5-H), 7.17(s, 1H,8-H), 3.94(s, 3H, —OCH₃), 3.91(s, 3H, —OCH₃). GC/MS m/z 456(M⁺+1,54.40),455(M⁺, 100.00), 454(M³⁰ −1,78.01), 439(M⁺ —OH, 7.96), 376(M⁺+1-Br,9.76), 375(M⁺-Br, 10.91), 360(5.23). Anal. (C₁₆H₁₃Br₂N₃O₃) C, H, N.

4-(3′-Bromo-4′-methylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P111]

yield 82.22%; m.p.225.0-228° C. ¹H NMR(DMSO-d₆): δ 10.23(s, 1H, —NH),8.62(s, 1H, 2-H), 8.06(D, 1h, j_(2′,6′)=2.1 Hz. 2′-H), 7.89(s, 1H, 5-H),7.71(dd, 1H, J_(5′,6′)=8.7 Hz, J_(2′,6′)=2.1 Hz, 6′-H), 7.37(d, 1H,J_(5′,6′)=8.7 Hz, 5′-H, 7.21(s, 1H, 8-H), 3.96(s, 3H, —OCH₃), 3.93(s,—OCH₃). UV(MeOH)λ_(max)(ε): 204.0, 228.0, 255.0, 320.0 nm.IR(KBr)υ_(max): 3431, 3248, 2835, 1633, 1517, 1441, 1281, 1155 cm⁻¹.GC/MS m/z 375(M⁺+1, 76.76), 374(M⁺, 100.00), 373(M³⁰ −1, 76.91),358(M⁺+1 —OH, 11.15), 357(1.42), 356(6.31). Anal. (C₁₇H₁₆BrN₃O₂HCl) C,H, N.

4-(4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P131]

yield 84.29%; m.p. 245.0-248.0.° C. UV(MeOH)λ_(max): 203.0, 222.0,251.0, 320.0 nm; IR(KBr)υ_(max): 3428, 2836, 1635, 1516, 1443, 1234cm⁻¹; ¹H NMR(DMSO-d₆): δ 11.21(s, 1H, —NH), 9.70(s, 1H, —OH), 8.74(s,1H, 2-H), 8.22(s, 1H, 5-H), 7.40(d, 2H, J=8.9 Hz, 2′,6′-H), 7.29(s, 1H,8-H), 6.85(d, 2H, J=8.9 Hz, 3′,5′-H), 3.98(s, 3H, —OCH₃), 3.97(s, 3H,—OCH₃). GC/MS m/z 298 (M⁺+1, 100.0), 297(M⁺, 26.56), 296(M⁺−1, 12.46).Anal. (C₁₆H₁₅N₃O₃HCl) C, H, N.

4-(2′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P132]

yield 82.49%; m.p. 255.0-258.0 ° C. ¹H NMR(DMSO-d₆): δ 9.78(s, 1H, —NH),9.29(s, 1H, —OH), 8.33(s, 1H, 2-H), 7.85(s, 1H, 5-H), 7.41-6.83(m, 4H,3′, 4′, 5′, 6′-H), 7.16(s, 1H, 8-H), 3.93(s, 3H, —OCH₃), 3.92(s, 3H,—OCH₃). UV(mEoh)λ_(max)(ε): 203.0, 224.0, 245.0, 335.0 nm.IR(KBr)υ_(max): 3500 (br), 3425, 2833, 1625, 1512, 1456, 1251, 1068cm⁻¹. GC/MS m/z 298(M⁺+1, 8.91), 297(M⁺, 56.64), 281(M⁺+1 -OH, 23.47),280(M⁺-OH, 100.00). Anal. (C₁₆H₁₅N₃O₃HCl) C, H, N.

4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P154]

yield 89.90%; m.p. 233.0-233.5° C. UV(MeOH)λ_(max): 203.0, 222.0, 250.0,335.0 nm; IR(KBr)υ_(max): 3431 br, 2841, 11624, 1498, 1423, 1244 cm³¹ ¹;¹H NMR(DMSO-d₆): δ 10.08(s, 1H, —NH), 9.38(s, 1H, —OH), 8.40(s, 1H, 2-H), 7.89(d, 1H, J_(2′, 5′)=2.7 Hz, 2′-H), 7.75(s, 1H, 5-H), 7.55(dd, 1H,J_(5′,6′)=9.0 Hz, J_(2′,6′)=2.7 Hz, 6′-H), 7.14(s, 1H, 8-H), 6.97(d, 1H,J_(5′,6′)=9.0 Hz, 5′-H), 3.92(s, 3H, —OCH₃), 3.90(s, 3H, —OCH₃). GC/MSm/z 378(M⁺+2, 90.68), 377(M⁺+1, 37.49), 376(M⁺, 100.00), 360(M⁺, 3.63),298(18.86), 282 (6.65). Anal. (C₁₆H₁₄N₃O₃HCl) C, H, N.

4-(3′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P180]

yield 71.55%; m.p. 256.0-258.0° C. ¹H NMR(DMSO-d₆): δ 9.41(s, 1H, —NH),9.36(s, 1H, —OH), 8.46(s, 1H, 2-H), 7.84(s, 1H, 5-H), 7.84-6.50(m, 4H,2′, 4′, 5′, 6′-H), 7.20(s, 1H, 8-H), 3.96(s, 3H, —OCH₃), 3.93(s, 3H,—OCH₃). UV(MeOH)λ_(max)(ε): 204.0, 224.0, 252.0, 335.0 nm.IR(KBr)υ_(max): 3394, 2836, 1626, 1508, 1429, 1251 cm¹. GM/MS m/z:297(M⁺, 61.89), 296(M⁺, 61.89), 296(M⁺−1, 100.00), 280(M⁺-OH, 13.63).Anal. (C₁₆H₁₅N₃O₃.HCl) C, H, N.

4-(3′-Chloro4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P971]

yield 84,.14%; m.p. 245.0° C.(dec). ¹H NMR(DMSO-d₆): δ 10.00(s, 1H,—NH), 9.37(s, 1H, —OH), 8.41(s, 1H, 2-H), 7.78(s,1H, 5-H), 7.49(d, 1H,J_(2′,6′)=2.7 Hz, 2′-H), 7.55(dd, 1H, J_(5′6′)9.0 Hz, J_(2′,6′)=2.7 Hz.,6′-H), 7.16(s, 1H, 8-H), 6.97(d, 1H, J_(5′,6′)=9.0 Hz, 5′-H), 3.93(s,3H, —OCH,), 3.91(s, 3H, —OCH₃). UV(MeOH)λ_(max)(ε): 209.0, 224.0, 249.0,330.0 nm. IR(KBr)υ_(max): 3448, 2842, 1623, 1506, 1423, 1241 cm⁻¹. GC/MSm/z: 341(M⁺, 100.00), 326(M⁺-CH₃, 98.50), 310(M⁺-OCH₃, 12.5), 295(9.0),189(13.5), 155(13.8). Anal.(C₁₆H₁₄CIN₃O₃.HCl) C, H, N.

4-(2′-Hydroxy-naphthyl-3′)-amino-6,7-dimethoxyquinazoline[WHI-P292].

Yield 87.41%; m.p.277.0-279.0° C., IR(KBr)υ_(max): 3479, 3386, 3036,2901, 1632, 1581, 1504, 1437, 1281 cm⁻¹. ¹H NMR (DMSO-d₆): δ 611.38(s,1H, —NH), 10.35(s, 1H, —OH), 8.73(s, 1H, 2-H), 8.25(s, 1H, 5-H),7.93-7.30(m,6H, 1′, 4′, 5′, 6′, 7′, 8′-H), 7.37(s, 1H, 8H), 4.00(s, 6H,—OCH₃). GC/MS m/a: 281(41.0), 253(11.0), 207(100.0). Anal.(C₂₀H₁₇N₃O₃.HCl) C, H, N.

Cytotoxic Compounds

As shown in the Example below, a hydroxyl substituent on the phenyl ring(R₁-R₅) appears to be necessary for cytotoxic effects of the novelsubstituted compounds of the invention, while a second substitution inthis ring, e.g., with a halogen such as bromine (Br), enhances thecytotoxic effect of the compound. In the method of the invention, thecytotoxic effects of these compounds is achieved by contacting braintumor cells with micromolar amounts of the inhibitory compound.Particularly useful cytotoxic compounds include WHI-P154 (3-Br, 4-OHsubstituted) and WHI-P131 (4-OH substituted), and WHI-P292 (1-OHnapthalene-substituted). More particularly useful are conjugates ofthese compounds with a targeting moiety such as EGF, having cytotoxicactivity at nanomolar concentrations.

As described above, compounds useful in the method of the invention alsoinclude those substituted with SH or NH₂ in place of the demonstratedhydroxy substitutions.

Compounds for Inhibiting Adhesion/Migration

The Examples below further demonstrate the effectiveness of thehydroxy-substituted quinazoline compounds of the invention as inhibitorsof brain tumor cell adhesion to extracellular matrix and of tumor cellmigration. Each of the tested compounds having a hydroxy substituents onthe phenyl ring demonstrated inhibitory activity against glioblastomacell adhesion/migration. Particularly potent and useful inhibitorycompounds include WHI-P154, WHI-P131, and WHI-P292.

Useful compounds of the invention are tested for the ability to preventadhesion/migration of brain tumor cells by assays described in theExamples below. Such assays include inhibition of cell blinding toextracellular matrix proteins in the presence of the inhibitory compoundas compared with a non-inhibitory control; inhibition of brain tumorcell invasion into Matrigel Matrix according to the method published byAlbini et.al., 1987, Cancer Res. 47:3239; and inhibition of focaladhesion plaques and actin polymerization in the presence of theinhibitory compound as compared with a non-inhibitory control.Conjugation of the inhibitory compounds to a targeting moiety, EGF,enhanced the inhibitory activity of the compound WHI-P154.

In the method of the invention, brain tumor cells are contacted withapproximately micromolar concentrations of the inhibitory compounds toinhibit tumor cell adhesion and invasion/migration into non-diseasedtissue. This is important, for example, during ablation surgery whencells may be dispersed. Adhesion of cells to ECM coupled with theaggressive malignant nature of brain tumor cells can result in new tumorgrowth at the adhesion site. Inhibition of adhesion and migration byadministering the compounds of the invention thereby inhibits new tumorgrowth.

Synthesis of Novel Hydroxy-Substituted Quinazoline Derivatives

The hydroxy-substituted quinazoline derivatives of the invention can besynthesized from a key starting material,4-chloro-6,7-dimethoxyquinazoline, prepared using published procedures(Nomoto, et al., 1990, Chem. Pharm. Bull., 38:1591-1595; Thomas, C. L.,1970, Academic Press, New York N.Y., “I. Synthesis of quinazolinederivatives”) as outlined below in Scheme 1 and as described more fullyin the Examples below:

The compounds of the invention are then prepared by the condensation of4-chloro-6,7-dimethoxyquinazoline with the appropriate substitutedaniline as outlined below in Scheme 2:

In a similar manner, compounds of the invention where X is S or O, orwhere X is alkyl, such as CH₂ are synthesized from a precursor compound,reacting the ring moiety with the desired substitution to produce thedesired product. These synthetic methods are know to those in the art,arid include, for example, those shown in Scheme 3 below.

Conjugates of the Invention

The term “conjugate” is meant to include a compound formed as acomposite between two or more molecules. More specifically, in thepresent invention, the novel hydroxy-substitutes quinazoline derivativesare bonded, for example, covalently bonded, to cell-specific targetingmoieties forming a conjugate compound for efficient and specificdelivery of the agent to a cell of interest.

Targeting Moiety

The phrase “targeting moiety” is meant to include a molecule whichserves to deliver the compounds of the invention to a specific site forthe desired activity. Targeting moieties include, for example, moleculesthat specifically bind molecules on a specific cell surface. Suchtargeting moieties useful in the invention include anti-cell surfaceantigen antibodies, growth factors which bind to cell surface receptorssuch as EGF and its receptor EGF-R. Cytokines, including interleukinsand factors such as granulocyte/macrophage stimulating factor (GMCSF)are also specific targeting moieties, known to bind to specific cellsexpressing high levels of their receptors.

Epidermal Growth Factor (EGF) and Its Receptor (EGF-Rc)

Human Epidermal Growth Factor (hEGF) is commercially available in ahighly purified form, for example, from Upstate Biotechnology, Inc.(Lake Placid, N.Y.) (Lot No. 01-107C). This protein ligand is known tobind specifically and with high affinity to receptors located on thesurface of EGF-responsive cells. Expression of the EFG-Rc is increasedin EGF-responsive cells, including hyperplastic neointima cells.

For use in the conjugates of the present invention, recombinant humanEGF (hrEGF) is preferred, although it is anticipated that hEGF and hEGFanalogs that specifically bind hEGF-Rc on neointima cells will similarlyinhibit migration of vascular cells and formation of hyperplasticneointima cell growth when conjugated.

Human epidermal growth factor (EGF) is a 53 amino acid, single-chain,6216 daltons polypeptide, which exerts biologic effects by binding to aspecific 170 kDa cell membrane epidermal growth factor receptor(EGF-receptor/ErbB-1) (Fix, S. B., 1994, Breast Cancer Research &Treatment, 29:41-49; Earp et at., 1995, Breast Cancer Research &Treatment, 35:115-132; Wright, et al., 1995, J. Biol. Chem.,270:12085-12093; Broome and Hunter, 1996, J. Biol. Chem,271:16798-16806). The human EGF-receptor consists of an extracellulardomain with a high cysteine content and N-linked glycosylation, a singletransmembrane domain, and a cytoplasmic domain with protein tyrosinekinase (PTK) activity.

Binding of EGF to the EGF-receptor/ErbB-1 results in receptordimerization with itself or other members of the Erb-B (subtype I)transmembrane PTK family (e.g., Erb-B2, Erb-B3), resulting in activationwith autophosphorylation of the PTK domain (Muthuswamy, S. K., 1994,Molecular & Cellular Biology, 14:735-743); Ottenhoff-Kalff et al., 1992,Cancer Research, 52:4773-4778). The EGF-receptor is physically andfunctionally associated with Src protooncogene family PTK, includingp60^(STC) (Muthuswamy, S. K., 1994, Molecular & Cellular Biology,14:735-743; Ottenhoff-Kalff et al., 1992, Cancer Research, 52:4773-4778;Aikyama et al., 1987, J. Biol. Chem., 262:5592-5595). This associationis believed to be an integral part of the signaling events mediated bythe EGF-receptor (Ottenhoff-Kalff et al., 1992, Cancer Research,52:4773-4778).

Conjugate Formation

To form the conjugates of the invention, targeting moieties arecovalently bonded to sites on the hydroxy-substituted quinazolinecompounds. The targeting moiety, which is often a polypeptide molecule,is bound to compounds of the invention at reactive sites, including NH₂,SH, CHO, COOH, and the like. Specific linking agents are used to linkthe compounds. Preferred linking agents are chosen according to thereactive site to which the targeting moiety is to be attached.

Methods for selecting an appropriate linking agent and reactive site forattachment of the targeting moiety to the compound of the invention areknown, and are described, for example, in Hermanson, et al.,Bioconjugate Techniques, Academic Press, 1996; Hermanson, et al.,Immobilized Affinity Ligand Techniques, Academic Press, 1992; and PierceCatalog and Handbook, 1996, pp. T155-T201. One exemplary method forconjugating EGF is described in the Examples below.

Administration Methods

The conjugates of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host,including a human patient in a variety of forms adapted to the chosenroute of administration and suitable for administration of the smallmolecule or its conjugate. Preferred administration routes includeorally, parenterally, as well as intravenous, intramuscular orsubcutaneous routes.

It is preferred that the conjugate of the present invention beadministered parenterally, i.e., intravenously or intraperitoneally, byinfusion or injection. In one embodiment of the invention, the compoundsmay be administered directly to a tumor by tumor injection; by injectingthe compound into the brain, e.g., into the ventricular fluid; or bysystemic delivery by intravenous injection. The compounds of theinvention, including the conjugates, are of a size and compositionexpected to have ready access to the brain across the blood-brainbarrier.

Solutions or suspensions of the conjugates can be prepared in water,isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.Dispersions may also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions or dispersions or sterile powderscomprising an active ingredient which are adapted forte extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.In all cases, the ultimate dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols and the like, vegetableoils, nontoxic glyceryl esters, and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. prevention of theaction of microorganisms can be accomplished by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like. In many cases, it will bedesirable to include isotonic agents, for example, sugars, buffers, orsodium chloride. Prolonged absorption of the injectable compositions canbe brought about by the inclusion in the composition of agents delayingabsorption—for example, aluminum monosterate hydrogels and gelatin.

Sterile injectable solutions are prepared by incorporating theconjugates in the required amount in the appropriate solvent withvarious other ingredients as enumerated above and, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying techniques, which yielda powder of the active ingredient plus any additional desired ingredientpresent in the previously sterile-filtered solutions.

Tumor Treatment

For purposes of this invention, a method of tumor treatment includescontacting brain tumor cells with a compound of the invention in orderto achieve an inhibition of tumor cell growth, a killing of tumor cells,and/or increased patient survival time. Treatment of tumors by themethod of the invention, also includes the prevention of the adhesionand migration of tumor cells, thereby inhibiting metastases.

The cytotoxic and adhesion/migration-inhibiting compounds of theinvention are suitable for use in mammals. As used herein, “mammals”means any class of higher vertebrates that nourish their young with milksecreted by mammary glands, including, for example, humans, rabbits, andmonkeys.

Apoptosis

Apoptosis, or programmed cellular death, is an active process requiringnew protein synthesis. Typically, the process requires ATP, involves newRNA and protein synthesis, and culminates in the activation ofendogenous endonucleases that degrade the DNA of the cell, therebydestroying the genetic template required for cellular homostasis.Apoptosis is observed in controlled deletion of cells duringmetamorphosis. differentiation, and general cell turnover and appearsnormally to be regulated by receptor-coupled events. For these reasons,apoptosis has been called “programmed cell death” or “cell suicide.”While every cell likely has the genetic program to commit suicide, it isusually suppressed. Under normal circumstances, only those cells nolonger required by the organism activate this self-destruction program.

Apoptotic cell death is characterized by plasma membrane blebbing, cellvolume loss, nuclear condensation, and endonucleolytic degradation ofDNA at nucleosome intervals. Loss of plasma membrane integrity is arelatively late event in apoptosis, unlike the form of cell death termednecrosis, which can be caused by hypoxia and exposure to certain toxinsand which is typically characterized early-on by increased membranepermeability and cell rupture.

Adhesion/Migration

Adhesion is meant to include that activity of a cell, such as a tumorcell, by which it adheres to extracellular matrix proteins, includinglaminin, fibronectin, and collagen. Adhesion assays are known, andinclude, for purposes of this invention, the adherence of tumor cells toplates coated with extracellular matrix proteins.

Migration is meant to include that activity of tumor cells by which theymigrate through extracellular matrix and invade tissues. Assays formigration include the ability of cells to migrate through a matrixformed of extracellular matrix, such as MATRIGEL matrix, as well asevaluation of the cell's cytoskeletal organization including actincytoskeletal rearrangement and changes in focal adhesions as describedin the following Examples.

Useful Dose

When used in vivo to selectively kill brain tumor cells or to inhibitbrain tumor cell adhesion/migration, the administered dose is thateffective to have the desired effect, e.g., sufficient to reduce oreliminate brain tumors, or sufficient to inhibit adherence/migration oftumor cells. Appropriate amounts can be determined by those skilled inthe art, extrapolating using known methods and relationships, from thein vitro and in vivo data provided in the Examples. Based on the SCIDmouse pharmacology data contained in this application, effectiveexposure levels are expected to be achieved.

In general, the dose of the novel substituted quinozalines effective toachieve brain tumor cell apoptosis, reduction in tumors, and increasedsurvival time, is hat which administers micromolar amounts of thecompound to the cells, preferably 100 micromolar or greater. Therequired dose is lessened by conjugation of the compound to a targetingmoiety, for example, to preferably 100 nanomolar or greaterconcentrations.

For cell adhesion and migration inhibitory activities, the compound isadministered generally at lower dosages, in the range of 100 micromolaror less.

The effective dose to be administered will vary with conditions specificto each patient. In general, factors such as the disease burden, tumorlocation (exposed or remote), host age, metabolism, sickness, priorexposure to drugs, and the like contribute to the expected effectivenessof a drug. One skilled in the art will use standard procedures andpatient analysis to calculate the appropriate dose, extrapolating fromthe data provided in the Examples.

In general, a dose which delivers about 1-100 mg/kg body weight isexpected to be effective, although more or less may be useful.

In addition, the compositions of the invention may be administered incombination with other anti-tumor therapies. In such combinationtherapy, the administered dose of the hydroxy-substituted quinazolinederivatives would be less than for single drug therapy.

EXAMPLES

The invention may be further clarified by reference to the followingExamples, which serve to exemplify some of the preferred embodiments,and not to limit the invention in any way.

Example 1 Synthesis of Quinazoline Derivatives

All chemicals were purchased from the Aldrich Chemical Company,Milwaukee, Wisconsin, and were used directly for synthesis. Anhydroussolvents such as acetonitrile, methanol, ethanol, ethyl acetate,tetrahydrofuran, chloroform, and methylene chloride were obtained fromAldrich as sure seal bottles under nitrogen and were transferred toreaction vessels by cannulation. All reactions were carried out under anitrogen atmosphere.

The key starting material, 4-chloro-6,7-dimethoxyquinazoline, wasprepared using published procedures (Nomoto, et al., 1990, Chem. Pharm.Bull., 38:1591-1595; Thomas, C. L., 1970, Academic Press, New York,N.Y., “I. Synthesis of quinazoline derivatives”) as outlined below inScheme 1:

Specifically, 4,5-dimethoxy-2-nitrobenzoic acid (compound 1) was treatedwith thionyl chloride to form acid chloride, followed by reacting withammonia to yield 4,5-dimethoxy-2-nitrobenzamide (compound 2). Compound 2was reduced with sodium borohydride in the presence of catalytic amountsof copper sulphate to give 4,5-dimethoxy-2-aminobenzamide (compound 3),which was directly refluxed with formic acid to yield6,7-dimethoxyquinazoline-4(3H)-one (compound 4). Compound 4 was refluxedwith phosphorus oxytrichloride to give 4-chloro-6,7-dimethoxyquinazoline(compound 5) in good yield.

Substituted quinazoline derivatives were prepared by the condensation of4-chloro-6,7-dimethoxyquinazoline with substituted anilines as outlinedbelow in Scheme 2:

Specifically, a mixture of 4-chloro-6,7-dimethoxyquinazoline (448 mg, 2mmols) and the substituted aniline (2.5 mmols) in EtOH (20 ml) washeated to reflux. After refluxing for 4-24 hours, an excess amount ofEt₃N was added, and the solvent was concentrated to give the crudeproduct which was recrystalized from DMF.

As discussed above, the novel hydroxy-substituted quinazolinederivatives of the invention were created by reacting substitutedanilines with the key starting material,4-chloro-6,7-dimethoxyquinazoline. Each of the anilines to synthesizethe compounds is shown in the table below.

forms WHI-P79 forms WHI-P131 forms WHI-P111 forms WHI-P180

forms WHI-P97 forms WHI-P132 forms WHI-P154 forms WHI-P197

forms WHI-P292 forms WHI-P258

Example 2 Characterization of Substituted Quinazoline Derivatives

The substituted quinazoline derivatives were synthesized as described inExample 1 and characterized. Each structure is shown below, along withits identifying analytical test results. Proton and carbon NuclearMagnetic Resonance (¹H and ¹³C NMR) spectra were recorded on a Mercury2000 Varian spectrometer operating at 300 MHz and 75 MHz, respectively,using an automatic broad band probe. Unless otherwise noted, all NMRspectra were recorded in CDCl₃ at room temperature. ¹H chemical shiftsare quoted in parts per million (δ in ppm) downfield from tetramethylsilane (TMS), which was used as an internal standard at 0 ppm and s, d,t, q, m designate singlet, doublet, triplet, quartet and multiplet,respectively. Melting points were determined using a Fisher-Johnsmelting apparatus and are uncorrected. UV spectra were recorded using aBeckmann Model # DU 7400 UV/V is spectrometer with a cell path length of1 cm. Methanol was used as the solvent for the UV spectra. FourierTransform Infrared spectra were recorded using an FT-Nicolet modelProtege #460 instrument. The infrared spectra of the liquid samples wererun as neat liquids using KBr discs. The KBr pellet method was used forall solid samples. The GC/mass spectrum analysis was conducted using aHewlett-Packard GC/mass spectrometer model #6890 equipped with a massion detector and Chem Station software. The temperature of the oven wassteadily increased from 70° C. to 250° C. and the carrier gas washelium.

4-(3′-Bromophenyl)-amino-6,7-dimethoxyquinazoline [WHI-P79]

yield 84.17%; m.p. 246.0-249.0° C. UV(MeOH)λ_(max): 217.0, 227.0, 252.0nm; IR(KBr)ν_(max): 3409, 2836, 1632, 1512, 1443, 1243, 1068 cm⁻¹; ¹HNMR(DMSO-d₆): δ 10.42(br, s, 1H, NH), 8.68(s, 1H, 2-H), 8.07-7.36(m, 5H,5,2′, 4′, 5′, 6′-H), 7.24(s, 1H), 8H), 3.98(s, 3H, —OCH₃), 3.73(s, 3H,—OCH₃); GC/MS m/z 361(M⁺ + 1, # 61.83), 360(M⁺, 100), 359(M⁺ − 1,63.52), 344(11.34), 222(10.87), 140(13.65). Anal. (C₁₆H₁₄BrN₃O₂) C, H,N. 4-(3′, 5′-Dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P97]

yield 72.80%; m.p. > 300.0° C. UV(MeOH)λ_(max): 208.0, 210.0, 245.0,320.0 nm; IR(KBr)υ_(max): 3504(br), 3419, 2868, 1627, 1512, 1425, 1250,1155 cm⁻¹; ¹H NMR(DMSO-d₆): δ 9.71(s, 1H, —NH), 9.39(s, 1H, —OH),8.48(s, 1H, 2-H), 8.07(s, 2H, 2′, 6′-H), 7.76(s, 1H, 5-H), 7.17(s, 1H,8-H), 3.94(s, 3H, —OCH₃), 3.91(s, 3H, # —OCH₃). GC/MS m/z 456(M⁺ + 1,54.40), 455(M⁺, 100.00), 454(M⁻− 1, 78.01), 439(M⁺ —OH, 7.96), 376(M⁺ +1-Br, 9.76), 375(M⁺ − Br, 10.91), 360(5.23). Anal. (C₁₆H₁₃Br₂N₃O₃) C, H,N. 4-(3′-Bromo-4′methylphenyl)-amino-6,7-dimethoxyquinazoline [WHI-P111]

yield 82.22%; m.p. 225.0-228° C. ¹H NMR(DMSO-d₆): δ 10.23(s, 1H, —NH),8.62(s, 1H, 2-H), 8.06(D, 1h, j_(2′,6′)=2.1 Hz. 2′-H), 7.89(s, 1H, 5-H),7.71(dd, 1H, J_(5′,6′)=8.7 Hz, J_(2′,6′)=2.1 Hz, 6′-H), 7.37(d, 1H,J_(5′,6′)=8.7 Hz, 5′-H, 7.21(s, 1H, 8-H), 3.96(s, 3H, —OCH₃), 3.93(s,—OCH₃). # UV(MeOH)λ_(max)(ε): 204.0, 228.0, 255.0, 320.0 nm.IR(KBr)υ_(max): 3431, 3248, 2835, 1633, 1517, 1441, 1281, 1155 cm⁻¹.GC/MS m/z 375(M⁺ + 1, 76.76), 374(M⁺, 100.00), 373(M⁺ − 1, 76.91),358(M⁺ + 1-OH, 11.15), 357(1.42), 356(6.31). Anal. (C₁₇H₁₆BrN₃O₂HCl) C,H, N. 4-(4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline [WHI-P131]

yield 84.29%; m.p. 245.0-248.0.° C. UV(MeOH)λ_(max): 203.0, 222.0,251.0, 320.0 nm; IR(KBr)υ_(max): 3428, 2836, 1635, 1516, 1443, 1234cm⁻¹; ¹H NMR(DMSO-d₆): δ 11.21(s, 1H, —NH), 9.70(s, 1H, —OH), 8.74(s,1H)2-H), 8.22(s, 1H, 5-H), 7.40(d, 2H, J=8.9 Hz, 2′, 6′-H), 7.29(s, 1H,8-H), 6.85(d, 2H, J=8.9 Hz, 3′, 5′-H), 3.98(s, 3H, #—OCH₃)3.97(s,3H,—)CH₃). GC/MS m/z 298(M⁺ + 1, 100.00), 297(M⁺, 26.56),296(M⁺− 1, 12.46). Anal. (C₁₆H₁₅N₃O₃HCl) C, H, N.4-(2′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline [WHI-P132]

yield 82.49%; m.p. 255.0-258.0° C. ¹H NMR(DMSO-d₆): δ 9.78(s, 1H, —NH),9.29(s, 1H, —OH), 8.33(s, 1H, 2-H), 7.85(s, 1H, 5-H), 7.41-6.83(m, 4H,3′, 4′, 5′, 6′-H), 7.16(s, 1H, 8-H), 3.93(s, 3H, —OCH₃), 3.92(s, 3H,—OCH₃). UV(mEoh)λ_(max)(ε): 203.0, 224.0, 245.0, 335.0 nm.IR(KBr)υ_(max): 3500(br), 3425, 2833, 1625, 1512, 1456, # 1251, 1068cm⁻¹. GC/MS m/z 298(M⁺ + 1, 8.91), 297(M⁺, 56.64), 281(M⁺ + 1-OH,23.47), 280(M⁺ − OH, 100.00). Anal. (C₁₆H₁₅N₃O₃HCl) C, H, N.4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline [WHI-P154]

yield 89.90%; m.p. 233.0-233.5° C. UV(MeOH)λ_(max): 203.0, 222.0, 250.0,335.0 nm; IR(KBr)υ_(max): 3431 br, 2841, 1624, 1498, 1423, 1244 cm⁻¹; ¹HNMR(DMSO- d₆): δ 10.09(s, 1H, —NH), 9.38(s, 1H, —OH), 8.40(s, 1H, 2-H),7.89(d, 1H, J_(2′, 5′)=2.7 Hz, 2′-H), 7.75(s, 1H, 5-H), # 7.55(dd, 1H,J_(5′,6′)=9.0 Hz, J_(2′,6′)=2.7 Hz, 6′-H), 7.14(s, 1H, 8-H), 6.97(s, 1H,J_(5′,6′)= 9.0 Hz, 5′H), 3.92(s, 3H, —OCH₃), 3.90(s, 3H, —OCH₃). GC/MSm/z 378(M⁺ + 2, 90.68), 377(M⁺ + 1, 37.49), 376(M⁺, 100.00), 360(M⁺,3.63), 298(18.86), 282(6.65). Anal. # (C₁₆H₁₄N₃O₃HCl) C, H, N.4-(3′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline [WHI-P180]

yield 71.55%; m.p. 256.0-258.0° C. ¹H NMR(DMSO- d₆): δ 9.41(s, 1H, —NH),9.36(s, 1H, —OH), 8.46(s, 1H, 2- H), 7.84(s, 1H, 5-H), 7.84-6.50(m, 4H,2′, 4′, 5′, 6′-H), 7.20(s, 1H, 8-H), 3.96(s, 3H, —OCH₃), 3.93(s, 3H,—OCH₃). UV(MeOH)λ_(max)(ε): 204.0, 224.0, 252.0, 335.0 nm. #IR(DBr)υ_(max): 3394, 2836, 1626, 1508, 1429, 1251 cm¹. GM/MS m/z:297(M⁺, 61.89), 296(M⁺, 61.89), 296(M⁺ − 1, 100.00), 280(M⁺ −OH, 13.63).Anal. (C₁₆H₁₅N₃O₃.HCl) C, H, N.4-(3′-Chloro-4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P197]

yield 84.14%; m.p. 245.0° C. (dec). ¹H NMR(DMSO-d₆): δ 10.00(s, 1H,—NH), 9.37(s, 1H, —OH), 8.41(s, 1H, 2-H), 7.78(s, 1H, 5-H), 7.49(s, 1H,J_(2′, 6′)=2.7Hz, 2′-H), 7.55(dd, 1H, J_(5′,6′)=0.0 Hz, J_(2′, 6′)=2.7Hz., 6′-H), 7.16(s, 1H, 8-H), 6.97(d, 1H, J_(5′,6′)=9.0 Hz, 5′-H),3.93(s, 3H, —OCH₃), # 3.91(s, 3H, —OCH₃). UV(MeOH)λ_(max)(ε): 209.0,224.0, 249.0, 330.0 nm. IR(KBr)υ_(max): 3448, 2842, 1623, 1506, 1423,1241 cm⁻¹. GC/MS m/z: 341(M⁺, 100.00), 326(M⁺ − CH₃, 98.50), 310(M⁺ −OCH₃, 12.5), 295(9.0), 189(13.5), 155(13.8). Anal. (C₁₆H₁₄CIN₃O₃.HCl) C,H, N. 4-(phenyl)-amino-6,7-dimethoxyquinazoline [WHI-P258]

yield 88.6%; m.p. 258.0-260.0° C. ¹H NMR(DMSO-d₆): δ 11.41(s, 1H, —NH),8.82(s, 1H, 2-H), 8.32(s, 1H, 5-H), 7.70-7.33(m, 5H, 2′, 3′, 4′, 5′,6′-H), 7.36(s, 1H, 8H), 4.02(s, 3H, —OCH₃), 4.00(s, 3H, —OCH₃), 4.00(s,3H, —OCH₃). UV(MeOH)λ_(max)(ε): 210.0, 234.0, 330.0 nm. #IR(KBr)υ_(max): 2852, 1627, 1509, 1434, 1248 cm⁻¹. GC/MS m/z 282(M⁺ + 1,10.50), 281(M⁺, 55.00), 280(M⁺ − 1, 100.00), 264(16.00), 207(8.50).Anal. (C₁₆H₁₅N₃O₂) C, H, N.4-(2′-Hydroxy-naphthyl-3′-amino-6,7-dimethoxyquinazoline [WHI-P292].

Yield 87.41%; m.p. 277.0-279.0° C., IR(KBr)δ_(max): 3479, 3386, 3036,2901, 1632, 1581, 1504, 1437, 1281 cm⁻¹. ¹H NMR(DMSO-d₆): δ 11.38(s, 1H,—NH), 10.35(s, 1H, —OH), 8.73(s, 1H, 2-H), 8.25(s,1H,5-H), 7.93-7.30(m,6H, 1′, 4′, 5′, 6′, 7′, 8′-H),7.37(s, 1H, 8H), 4.00(s, 6H, —OCH₃). GC/MSm/a: 281(41.0), 253(11.0), 207(100.0). Anal. (C₂₀H₁₇N₃O₃.HCl) C, H, N.

Example 3 Cytotoxicity of Substituted Quinazoline Derivatives

The cytotoxicity of the substituted quinazoline derivative compoundsagainst human glioblastoma cells was evaluated. The relative importanceof particular substituent group on the compounds was also studied. Thesubstituted quinazoline derivative compounds, prepared as describedabove for Example 1, were tested, along with DMSO and Genistein ascontrols.

Cytotoxicity Assay

The cytotoxicity assay of various compounds against human brain tumorcell lines was performed using the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay(Boehringer Mannheim Corp., Indianapolis, Ind.). Briefly, exponentiallygrowing brain tumor cells were seeded into a 96-well plate at a densityof 2.5×10⁴ cells/well and incubated for 36 hours at 37° C. prior to drugexposure. On the day of treatment, culture medium was carefullyaspirated from the wells and replaced with fresh medium containing thequinazoline compounds WHI-P79, WHI-P97, WHI-P111, WHI-P131, WHI-P132,WHI-P154, WHI-P180, WHI-P197, WHI-P258, unconjugated EGF, or EGF-P154,as well as the tyrosine kinase inhibitory isoflavone genistein (GEN) atconcentrations ranging from 0.1 to 250 μM. Triplicate wells were usedfor each treatment.

Human glioblastoma cell line U373 was obtained from American. TypeCulture Collection (Rockville, Md.) and maintained as a continuous cellline in Dulbecco's modified Eagles's medium supplemented with 10% fetalbovine serum and antibiotics. The B-lineage acute lymphoblastic leukemiacell line Nalm-6 was used as a negative control.

The cells were incubated with the various compounds for 24-36 hours at37° C. in a humidified 5% CO₂ atmosphere. To each well, 10 μl of MTT(0.5 mg/ml final concentration) was added and the plates were incubatedat 37° C. for 4 hours to allow MTT to form formazan crystals by reactingwith metabolically active cells. The formazan crystals were solubilizedovernight at 37° C. in a solution containing 10% SDS in 0.01 M HCl. Theabsorbence of each well was measured in a microplate reader (Labsystems)at 540 nm and a reference wavelength of 690 nm. To translate the OD₅₄₀values into the number of live cells in each well, the OD₅₄₀ values werecompared to those on standard OD₅₄₀—versus—cell number curves generatedfor each cell line. The percent survival was calculated using theformula:$\text{\%~~Survival} = {\frac{\text{live cell number~~[test]}}{\text{live cell number~~[control]}}100}$

The IC50 values were calculated by non linear regression analysis.

In Situ Detection of Apoptosis

The demonstration of apoptosis was performed by the in situnick-end-labeling method using ApopTag in situ detection kit (Oncor,Gaithersburg, Md.) according to the manufacturer's recommendations.Exponentially growing cells were seeded in 6-well tissue culture platesat a density of 50×10⁴ cells/well and cultured for 36 hours at 37° C. ina humidified 5% CO₂ atmosphere. The supernatant culture medium wascarefully aspirated and replaced with fresh medium containingunconjugated EGF or EGF-P154 at a concentration of 10, 25 or 50 μg/ml.After a 36 hour incubation at 37° C. in a humidified 5% CO₂ incubator,the supernatants were carefully aspirated and the cells were treated for1-2 minutes with 0.1% trypsin. The detached cells were collected into a15 ml centrifuge tube, washed with medium and pelleted by centrifugationat 1000 rpm for 5 minutes. Cells were resuspended in 50 μl of PBS,transferred to poly-L-lysine coated coverslips and allowed to attach for15 minutes. The cells were washed once with PBS and incubated withequilibration buffer for 10 minutes at room temperature.

After removal of the equilibration buffer, cells were incubated for 1hour at 37° C. with the reaction mixture containing terminaldeoxynucleotidyl transferase (TdT) and digoxigenin-11-UTP for labelingof exposed 3′-hydroxyl ends of fragmented nuclear DNA. The cells werewashed with PBS and incubated with anti-digoxigenin antibody conjugatedto FITC for 1 hour at room temperature to detect the incorporated dUTP.After washing the cells with PBS, the coverslips were mounted ontoslides with Vectashield containing propidium iodide (Vector Labs,Burlingame, Calif.) and viewed with a confocal laser scanningmicroscope. Non-apoptotic cells do not incorporate significant amountsof dUTP due to lack of exposed 3-hydroxyl ends, and consequently havemuch less fluorescence than apoptotic cells which have an abundance ofexposed 3′-hydroxyl ends. In control reactions, the TdT enzyme wasomitted from the reaction mixture.

Results

The identity of the specific substituents on each aniline moiety aresummarized below:

Quinazoline Derivatives Identity of Substituents

WHI-P79 WHI-P97 WHI-P111 WHI-P131 WHI-P132 WHI-P154 WHI-P180 WHI-P197WHI-258 WHI-292 3-Br 3-Br, 5-Br, 4-OH 3-Br, 4-CH₃ 4-OH 2-OH 3-Br, 4-OH3-OH 3-Cl, 4-OH H 1-OH Naphthyl

The results of the cytotoxicity assay against brain tumor cells areshown in FIGS. 1A and 1B. In summary, those compounds having hydroxysubstitutions on the phenyl ring, were effective in killing brain tumorcells. The unsubstituted compound P258 and the 3-Br-substituted potentTK inhibitor P79 were ineffective in killing brain tumor cells.Genistein and DMSO controls were also ineffective.

Those substituted quinazoline derivatives having an hydroxyl group onthe aniline moiety demonstrated cytotoxic activity. Four compoundstested possessed a single hydroxyl group; at position 4 (WHI-P131), atposition 2 (WHI-P132), at position 3 (WHI-P180), and at position 1(WHI-P292). All four exhibited significant cytotoxicity, with theWHI-P180 (3-OH) compound demonstrating slightly stronger effects thanthe other two.

Compounds were having both a hydroxyl substituent and a halogensubstituents were also potent cytotoxic agents. The structure ofWHI-P197 (3-Cl, 4-OH) differs from that of WHI-P131(4-OH) only in thechlorine atom at position 3. As shown in FIGS. 1A and 1B, addition ofthe chlorine atom did not effect the cytotoxicity of this compound.

The structure of WHI-P154 (3-Br, 4-OH) differs from WHI-P131 (4-OH),only in the bromine atom at position 3. As shown in FIGS. 1A and 1B, theaddition of the bromine atom to this compound significantly increasedthe cytotoxicity of the compound.

The structure of WHI-P97 differs from that of WHI-P154 only in theadditional bromine atom added at position 5. FIG. 1 shows that there isessentially no benefit from the added second bromine atom.

WHI-P154, 4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazolineexhibited significant cytotoxicity against the U373 human glioblastomacell line in 3 of 3 independent experiments with a mean (±SE) IC50 valueof 167.4±26.9 μM and a composite survival curve IC50 value of 158.5 μM.In contrast, WHI-P79, a potent inhibitor of EGF-R quid Src familytyrosine kinases (Bos, et al., 1997, Clin. Cancer Res. 3:2099-2106 Fry,et al., 1994, Science (Washington, D.C.) 265:1093-1095) failed to causeany detectable cytotoxicity to U373 glioblastoma cells. Thus, thecytotoxicity of WHI-P154 to U373 cells cannot be explained by itstyrosine kinase inhibitory properties. This notion was further supportedby the inability of the PTK inhibitor genistein (included as controls)to cause detectable cytotoxicity to U373 cells (IC50 value >250 μM; FIG.1A).

Example 4 Enhanced Cytotoxicity of Conjugated WHI-P154 Against HumanGlioblastoma Cells

In contrast to normal glial cells and neurons, significant numbers ofglioblastoma cells express the EGF receptor (EGF-R) at high levels.Therefore, the EGF-R is a potential target for delivering cytotoxicagents to glioblastoma cells with greater efficiency (Mendelsohn, J. andBaselga, J., 1995, Biologic Therapy of Cancer: Principles and Practice,pp. 607-23).

Expression of EGF-R by Glioblastoma Cells

Surface expression of the EGF-R on the U373 and U87 human glioblastomacell lines (obtained and maintained as described for Example 3) wasconfirmed with immunofluorescence and confocal laser scanning microscopyusing monoclonal antibodies to the extracellular domain of the EGF-R.Immunofluorescence staining with anti-a-tubulin antibody and the nucleardye Toto-3 was used in combination with confocal laser scanningmicroscopy to examine the morphological features of U373 glioma cellstreated with either unconjugated EGF or EGF-P154. Cells were fixed inparaformaldehyde, immunostained with monoclonal antibody to EGF-R (greenfluorescence) and counterstained with TOTO-3 (blue fluorescence) Theimmunostained cells were analyzed with a laser scanning confocalmicroscope. Blue fluorescence represents nuclei. Both cell lines showeda diffuse granular immunoreactivity with the anti-EGF-R antibody (seeFIGS. 2A and 2B).

Preparation of EGF-P154 Conjugate

In an attempt to enhance the demonstrated anti-tumor activity of4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154)against glioblastoma cells, by improving its targeting to and cellularuptake by glioblastoma cells, the compound was conjugated to recombinanthuman EGF, as described below.

Recombinant human EGF (rhEGF) was produced in E. coli harboring agenetically engineered plasmid that contains a synthetic gene for humanEGF fused at the N-terminus to a hexapeptide leader sequence for optimalprotein expression and folding. The rhEGF fusion protein precipitated inthe form of inclusion bodies and the mature protein was recovered bytrypsin-cleavage followed by purification using ion exchangechromatography and HPLC. The recovered rhEGF was 99% pure byreverse-phase HPLC and SDS-PAGE with an isoelectric point of 4.6±0.2.The endotoxin level was 0.172 EU/mg.

The recently published photochemical conjugation method using thehetero-bifunctional photoreactive crosslinking agent, Sulfosuccinimidyl6-[4′azido-2′-nitrophenylamino]hexanoate (Sulfo-SANPAH) (Pierce ChemicalCo., Rockford, Ill.) was employed for the synthesis of the EGF-P154conjugate, as described in Uckun, et al., 1995, Science, 267:886-891.Sulfo-SANPAH modified rhEGF was mixed with a 10:1 molar ratio ofWHI-P154 [50 mM solution in dimethyl sulfoxide (DMSO)] and thenirradiated with gentle mixing for 10 minutes with UV light atwavelengths 254-366 nm with a multiband UV light-emitter (Model UVGL-15Mineralight; UVP, San Gabriel, Calif.). Photolytic generation of areactive singlet nitrene on the other terminus of EGF-SANPAH in thepresence of a 10-fold molar excess of WHI-P154 resulted in theattachment of WHI-P154 to EGF.

Excess WHI-P154 in the reaction mixture was removed by passage through apre-packed PD-10 column, and 12 kDa EGF-EGF homoconjugates with orwithout conjugated WHI-P154 as well as higher molecular weight reactionproducts were removed by size-exclusion high-performance liquidchromatography (HPLC). Reverse phase HPLC using a Hewlett-Packard (HP)1100 series HPLC instrument was used for separation of EGF-P154 fromEGF-SANPAH. After the final purification, analytical HPLC was performedusing a Spherisorb ODS-2 reverse phase column (250×4 mm,Hewlett-Packard, Cat. #79992402-584). Prior to the HPLC runs, a BeckmanDU 7400 spectrophotometer was used to generate a UV spectrum for each ofthe samples to ascertain the λmax for EGF-P154, EGF-SANPAH, andunmodified EGF.

Each HPLC chromatogram was subsequently run at wavelengths of 214, 265,and 480 nm using the multiple wavelength detector option supplied withthe instrument to ensure optimal detection of the individual peaks inthe chromatogram. Analysis was achieved using a gradient flow consistingof 0% to 100% eluent in a time interval of 0 to 30 minutes. Five μLsamples applied to the above column were run using the followinggradient program: 0-5 minutes: 0-20% eluent; 5-20 minutes: 20-100%eluent; 25-30 minutes: 100% eluent; and 30-35 minutes: 100-0% eluent.The eluent was a mixture of 80% acetonitrile(CH3CN), 20% H₂O and 0.1%TFA. Electrospray ionization mass spectrometry (Feng, et al., 1991, J.Am. Soc. Mass Spectrometry 2:387-401; Covey, et al., 1988, RapidCommunications in Mass Spectrometry 2:249-256) was performed sing a PESCIEX API triple quadruple mass spectrometer (Norwalk, Conn.) todetermine the stoichiometry of P154 and EGF in EGF-P154.

Uptake and Internalization of EGF-P154

The kinetics of uptake and cytotoxicity of the EGF-P154 conjugate inU373 glioblastoma cells were analyzed using immunofluorescence andconfocal laser microscopy for following the internalized EGF-R andEGF-P154 molecules, as well as morphologic changes in treated cells.

Immunofluorescence was used to (i) examine the surface expression ofEGF-receptor (EGF-R) on brain tumor cells, (ii) evaluate the uptake ofEGF-P154 by brain tumor cells and (iii) examine the morphologic featuresof EGF-P154 treated brain tumor cells. For analysis of EGF-R expressionand cellular uptake of EGF-P154, U87 and U373 glioblastoma cells wereplated on poly-L-lysine coated glass-bottom 35 mm Petri dishes (MattekCorp., Ashland, Mass.) and maintained for 48 hours. In uptake studies,the culture medium. was replaced with fresh medium containing 5 μg/mlEGF, EGF-P154 or unconjugated WHI-P154 and cells were incubated at 37°C. for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, and 24hours.

At the end of the incubation, cells were washed twice with PBS and fixedin 2% paraformaldehyde. The cells were permeabilized and non-specificbinding sites were blocked with 2.5% BSA in PBS containing 0.1% TritonX-100 for 30 minutes. To detect the EGF-R/EGF-P154 complexes, cells wereincubated with a mixture of a monoclonal antibody (1:10 dilution in PBScontaining BSA and Triton X-100) directed to the extracellular domain ofthe human EGF-R (Santa Cruz Biotechnologies Inc. Santa Cruz, Calif.) anda polyclonal rabbit anti-P154 antibody (1:500 dilution) for 1 hour atroom temperature. After rinsing with PBS, cells were incubated for 1hour with a mixture of a goat anti-mouse IgG antibody conjugated to FITC(Amersham Corp., Arlington Heights, Ill.) and donkey anti-rabbit IgGconjugated to Texas Red (Amersham Corp.) at a dilution of 1:40 in PBS.Similarly, tubulin expression was examined by immunofluorescence using amonoclonal antibody against α-tubulin (Sigma Chemical Co, St. Louis,Mo.) at a dilution of 1:1000 and an anti-mouse IgG conjugated to FITC.Cells were washed in PBS and counterstained with toto-3 (MolecularProbes Inc., Eugene, Oreg.) for 10 minutes at a dilution of 1:1000.Cells were washed again with PBS and the coverslips were mounted withVectashield (Vector Labs, Burlingame, Calif.) and viewed with a confocalmicroscope (Bio-Rad MRC 1024) mounted in a Nikon Labhophot uprightmicroscope.

Cytotoxic Activity

To determine if the improved delivery of WHI-P154 to glioblastoma cellsby conjugation to EGF resulted in potentiation of its anti-tumoractivity, the cytotoxic activities of EGF-P154 and unconjugated WHI-P154against U373 and U87 human glioblastoma cell lines were analyzed in doseresponse studies using in vitro MTT assays, as described above forExample 3. Apoptosis was also evaluated using the nick-end-labelingassay, as described for Example 3. The data are discussed below.

Results

Surface expression of the EGF-R on the U373 and U87 human glioblastomacell lines was confirmed with immunofluorescence and confocal laserscanning microscopy using monoclonal antibodies to the extracellulardomain of the EGF-R. As shown in FIGS. 2a and 2 b, both cell linesshowed a diffuse granular immunoreactivity with the anti-EGF-R antibody.

The kinetics of uptake and cytotoxicity of the EGF-P154 conjugate inU373 glioblastoma cells were analyzed using immunofluorescence andconfocal laser microscopy for following the internalized EGF-R andEGF-P154 molecules, as well as morphologic changes in treated cells.EGF-P154, similar to unconjugated EGF (not shown), was able to bind toand enter target glioblastoma cells via receptor-mediated endocytosis byinducing internalization of the EGF-R molecules. As shown in FIGS. 3A-C,3C′, 3D and 3D′, within 10 minutes after exposure to EGF-P154, theEGF-R/EGF-P154 complexes began being internalized, as determined byco-localization of the EGF-R (detected by anti-EGF-R antibody, greenfluorescence) and EGF-P154 (detected by anti-P154 antibody, redfluorescence) in the cytoplasm of treated cells. By 30 minutes, theinternalized EGF-R/EGF-P154 complexes were detected in the perinuclearregion of the treated glioblastoma cells. In contrast, cells treatedwith unconjugated WHI-P154 alone (FIGS. 3B, 3B′) did not reveal anydetectable redistribution of the surface EGF-R or cytoplasmic stainingwith the anti-P154 antibody (red fluorescence). By 24 hours (but not at6 or 12 hours), WHI-P154 molecules could also be detected in cellstreated with unconjugated WHI-154 (FIGS. 4A, 4A′, 4B, 4B′). Thus,conjugation of WHI-P154 to EGF resulted in increased uptake of thiscytotoxic quinazoline derivative by EGF-R positive glioblastoma cells.

To determine if the improved delivery of WHI-P154 to glioblastoma cellsby conjugation to EGF resulted in potentiation of its anti-tumoractivity, the cytotoxic activities of EGF-P154 and unconjugated WHI-P154against U373 and U87 human glioblastoma cell lines were analyzed in doseresponse studies using in vitro MTT assays, as described above forExample 3. As shown in FIG. 5A, EGF-P154 killed these glioblastoma cellsin each of 3 independent experiments at nanomolar concentrations withmean IC50 values of 813±139 nM (Range: 588-950 nM) for U373 cells and620±97 nM (Range: 487-761 nM) for U87 cells. The IC50 values derivedfrom the composite cell survival curves were 881 nM for U373 cells and601 nM for U87 cells. By comparison, unconjugated WHI-P154 killed U373or U87 cells only at micromolar concentrations. EGF-P154 was 206-foldmore potent than unconjugated WHI-P154 against U373 cells (IC50 values:167.4±26.9 μM vs 811±139 nM, P<0.003) and 288-fold more potent thanunconjugated WHI-P154 against U87 cells (IC50 values: 178.62±18.46 μM vs620±97 nM, P<0.001) (FIG. 5A).

Unlike WHI-P154, which showed marked cytotoxicity against the EGF-Rnegative NALM-6 leukemia cells, EGF-P154 elicited selective cytotoxicityto EGF-R positive glioblastoma cell lines only (FIGS. 5A and 5B). Thus,conjugation to EGF increased the potency of WHI-P154 against humanglioblastoma and at the same time restricted its cytotoxicity to EGF-Rpositive targets.

Unlike the EGF-P154 conjugate, BGF-GEN, a potent inhibitor of the EGF-Rtyrosine kinase and EGF-R associated Src family PTK, failed to killglioblastoma cells (FIG. 5A). Thus, the potent cytotoxicity of EGF-P154cannot be explained by the tyrosine kinase inhibitory properties of itsWHI-P154 moiety.

Immunofluorescence staining with anti-α-tubulin antibody and the nucleardye TOTO-3 was used in combination with confocal laser scanningmicroscopy to examine the morphological features of U373 glioma cellstreated with either unconjugated EGF or EGF-P154. As shown in FIGS.6A-6C, after 24 hours of exposure to 25 μg/ml EGF-P154 (but not 25 μg/mlunconjugated EGF), most of the glioma cells showed an abnormalarchitecture with complete disruption of microtubules, marked shrinkage,nuclear fragmentation and inability to adhere to the substratum. Thesemorphologic changes in EGF-P154-treated glioma cells were consistentwith apoptosis.

To confirm apoptotic DNA fragmentation in the nuclei of EGF-P154-treatedglioblastoma cells, an in situ apoptosis assay which allows thedetection of exposed 3′-hydroxyl groups in fragmented DNA byTdT-mediated dUTP nick-end labeling was used. As evidenced by theconfocal laser scanning microscopy images depicted in FIGS. 6D and 6E,EGF-P154 treated (but not EGF-treated) glioma cells examined fordigoxigenin-dUTP incorporation using FITC-conjugated anti-digoxigenin(green fluorescence) and propidium iodide counterstaining (redfluorescence) showed many apoptotic yellow nuclei with superimposedgreen and red fluorescence at 36 hours after treatment.

In summary, these data demonstrate specific and enhanced cytotoxicactivity of P154 when targeted to brain tumor cells by conjugation toEGF.

Example 5 Anti-tumor Activity of EGF-P154 in a SCID Mouse Model

The anti-tumor effects of conjugated EGF-P154 in vivo is demonstrated ina SCID mouse model. CB.17 SCID mice developed rapidly growing tumorsafter subcutaneous inoculation of 0.5×10⁶ U373 cells. The in vivoanti-tumor activity of EGF-P154 in this SCID mouse xenograft model ofhuman glioblastoma multiforme was examined.

Maintenance of SCID Mouse Colony

The SCID mice were housed in a specific pathogen-free room located in asecure indoor facility with controlled temperature, humidity, and noiselevels. The SCID mice were housed in microisolater cages which wereautoclaved with rodent chow. Water was also autoclaved and supplementedwith trimethoprim/sulfomethoxazol 3 days/week.

SCID Mouse Xenograft Model of Human Glioblastoma

The right hind legs of the CB. 17 SCID mice were inoculatedsubcutaneously (s.c.) with 0.5×10⁶ U373 human glioblastoma cells in 0.2mL PBS. SCID mice challenged with brain tumor cells were treated withEGF-P154 (0.5 mg/kg/dose or 1 mg/kg/dose in 0.2 ml PUS) as daily i.p.doses for 10 treatment days starting the day after inoculation of theglioblastoma cells. Daily treatments with PBS, unconjugated EGF (1mg/kg/dose), and unconjugated WHI-P154 (1 mg/kg/dose) were used ascontrols. Mice were monitored daily for health status and tumor growth,and were sacrificed if they became moribund developed tumors whichimpeded their ability to attain food or water, at the end of the 3-monthobservation period. Tumors were measured using Vernier calipers twiceweekly, and the tumor volumes were calculated according to the followingformula (Friedman, S. H., mDolan, M. E., Pegg, A. E., Marcelli, S.,Keir, S., Catino, J. J., Binger, D. D., Schold, S. C., Jr., 1995, CancerRes., 55:2853-2857): ${Volume} = \frac{{Width}^{2}*{Length}}{2}$

For histopathologic studies, tissues were fixed in 10% neutral bufferedformalin, dehydrated, and embedded in paraffin by routine methods. Glassslides with affixed 6 micron tissue sections were prepared and stainedwith hematoxylin/eosin. Primary endpoints of interest were tumor growthand tumor-free survival outcome. Estimation of life table outcome andcomparisons of outcome between groups were done, as previously reported(Waurzyniak, et al., 1997, Clinical Cancer Research 3:881-890; Anderson,et al., 1995, Cancer Res. 55:1321-1327; Uckun, et al., 1997, J. Clin.Oncol. 15:2214-2221).

Results

The conjugated quinazoline EGF-P154 significantly improved tumor-freesurvival in a dose-dependent fashion, when it was administered 24 hoursafter inoculation of tumor cells. FIGS. 7A and 7B show the tumor growthand tumor-free survival outcome of SCID mice treated with EGF-P154 (500μg/kg/day×10 days or 1 mg/kg/day×10 days), unconjugated EGF (1mg/kg/day×10 days), unconjugated WHI-P154 (1 mg/kg/day×10 days), or PBSafter inoculation with U373 glioblastoma cells.

None of the 15 control mice treated with PBS (N=5; median tumor-freesurvival=19 days), EGF (N=5; median tumor-free survival=23 days), orunconjugated WHI-P154 (N=5; median tumor-free survival=19 days) remainedalive tumor-free beyond 33 days (median tumor-free survival=19 days)(FIG. 7A). All of the 5 mice treated with EGF-P154 at the 500 μg/kg/daydose level developed tumors within 40 days with an improved mediantumor-free survival of 33 days (FIG. 7B). These tumors were much smallerthan these in control mice (FIG. 7A).

Tumors reached a size of 50 mm³ by 37.5±3.3 days in PBS treated mice,34.0±3.0 days in EGF-treated mice, and 36.0±5.1 days in WHI-P154 treatedmice. Tumors developing in EGF-P154 (500 μg/kg/day×10 days)-treated micereached the 50 mm³ tumor size approximately 11 days later than thetumors in control mice treated with PBS, EGF, or WHI-P154 (47.4±7.1 daysvs 35.8±1.8 days). The average size (mean±SE) of tumors at 20 days and40 days were 10.2±1.4 mm³ and 92.3±6.0 mm³. respectively for mice in thecontrol groups (i.e., PBS+EGF groups combined). By comparison, theaverage size (mean±SE) of tumors at 20 days and 40 days weresignificantly smaller at 1.0±1.1 mm³ (P=0.002) and 37.6±10.7 mm³(P=0.0003) for mice treated with EGF-P154 at the 500 μg/kg/day doselevel.

Notably, 40% of mice treated for 10 consecutive days with 1 mg/kg/dayEGF-P154 remained alive and free of detectable tumors for >58 days(PBS+EGF+WHI-P154 vs EGF-P154, P<0.00001 by log-rank test). The tumorsdeveloping in the remaining 60% of the mice did not reach a size >50 mm³during the 58-day observation period. Thus, EGF-P54 elicited significantin vivo anti-tumor activity at the applied nontoxic dose levels. Theinability of 1 mg/kg/day×10 days of unconjugated WHI-P154 (53.2 nmol)and unconjugated EGF to confer tumor-free survival in this SCID mousemodel in contrast to the potency of 1 mg/kg/day×10 days EGF-P154(corresponding to 2.9 nmol of WHI-P154) demonstrates that the in vivoanti-tumor activity of EGF-P154 cannot be attributed to its EGF moietyalone and also that conjugation to EGF enhances the in vivo anti-tumoractivity of WHI-P154 against glioblastoma cells by >8-fold.

Taken together, the findings of Examples 3-5 provide unprecedentedevidence that the substituted quinazoline3-bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154)exhibits significant cytotoxicity against human glioblastoma cells andthat its anti-tumor activity can be substantially enhanced byconjugation to EGF as a targeting molecule. Although WHI-P154 is apotent inhibitor of the EGF-R kinase as well as Src family tyrosinekinases, its cytotoxicity in glioblastoma cells cannot be attributed toits tyrosine kinase inhibitory properties alone, since4-(3′-Bromophenyl)-amino-6,7-dimethoxyquinazoline (WHI-P79) with equallypotent PTK inhibitory activity, failed to kill WHI-P154 sensitiveglioblastoma cells. Similarly, several PTK inhibitors capable of killinghuman leukemia and breast cancer cells lacked detectable cytotoxicityagainst glioblastoma cells. Glioblastoma cells exposed to EGF-conjugatedWHI-P154 underwent apoptosis. Although EGF was used to target WHI-P154to glioblastoma cells in the present study, other biologic agentsincluding different cytokines such as IGF and antibodies reactive withglioblastoma-associated antigens are also ted to be effective targetingmolecules for this novel quinazoline derivative.

Example 6 Substituted Quinazolines Inhibit Glioblastoma Cell Adhesion

During the multistep process of tissue invasion, tumor cells initiallyadhere to the extracellular matrix proteins via cell surface integrinreceptors and then gain migratory capacity to enter the surroundingtissues. ECM proteins such as laminin, fibronectin, and type IV collagenare thought to play an important role in tumor cell attachment andmigration. Laminin, fibronectin and collagen have been found in thebasal lamina of blood vessels and in the glial limitans externa in thebrain that promote he adhesion and invasion of tumor cells in situ(Carbonetto, S., 1984, Trends Neurosci., 7:382-387; Rutka, J. T.,Apodaca, G Stern, R., J. Neurosurg., 69:155-170; Venstrom, K. A. andReichard, L. F., 1993, FASEBJ., 7:996-1003). The effects of these ECMproteins on integrin-mediated glioblastoma cell adhesion was examinedusing four different human glioblastoma (U87, U373, T98, and U138) andone medulloblastoma (Daoy) cell line.

Cell Lines

Human brain tumor cell lines derived from adult patients withglioblastoma, U-87 MG (Cat. #HTB-14), U-118 MG (Cat. #HTB-15), U-138 MG(Cat. #HTB 16), U-373 MG (Cat. #HTB-17), T98-G (Cat. #CRL-1690) andmedulloblastoma Daoy (Cat. #HTB-186) were obtained from American TypeCulture Collection (ATCC, Rockville, Md.) and maintained in liquidculture using DMEM supplemented with 10% fetal bovine serum andantibiotics. Fibroblast conditioned medium was used as a source ofchemoattractant in vitro invasion assays Conditioned medium was preparedas described previously (Albini, A., Iwamto, Y., Kleinman, H. K.,Martin, G. R., Aaronson, S. A., Kozlowski, J. M., and MeEwan, R. N.,1987, Cancer Res., 47:3239-3245). For the preparation of thisconditioned medium NIH/3T3 embryonic fibroblasts (AATCC cat. #CRL-1658)were grown to 80% confluency in DMEM medium supplemented with 10% FBSand cultured for 24 hours in serum-free medium containing 0.5 μg/mlbovine serum albuminutes The culture supernatants were collected,centrifuged at 1000×g for 15 minutes to remove cellular debris and usedas conditioned medium.

Adhesion Assays

In vitro adhesion assays were performed to (a) study the baselineadhesive properties of various glioblastoma cell lines and (b) evaluatethe effects of quinazoline derivatives on the adhesive properties ofglioblastoma cells. The plates for the adhesion assays were precoatedwith the extracellular matrix proteins laminin, fibronectin or type IVcollagen (each at a final concentration of 1 μg/ml in PBS) overnight at4° C. and dried. On the day of the experiment, the wells were rehydratedand blocked with 10% bovine serum albumin in PBS for 1 hour at roomtemperature and used for the adhesion assays, as described below.

To study the effects of quinazoline derivatives on glioblastoma celladhesion, exponentially growing cells in DMEM were incubated with thecompounds WHI-P79, WHI-P97, WHI-P31, WHI-P154, WHI-P258 or genistein atconcentrations ranging from 1 μM to 100 μM for 16 hours in a humidified5% CO₂ atmosphere. DMSO (0.1%) was included as a vehicle control. Aftertreatment, cells were detached from the flasks with 0.05% trypsin (LifeTechnologies) resuspended in DMEM, incubated at 37° C. for 2 hours toallow them to recover from the trypsinization stress and examined fortheir ability to adhere to plates precoated with ECM proteins.

In adhesion assays, cells were centrifuged, washed twice with serum-freeDMEM, counted and resuspended in serum-free DMEM to a finalconcentration of 2.5×10⁵ cells/ml. One hundred μl of the cell suspensioncontaining 2.5×10⁴ cells were added to each well and cells were allow dto adhere for 1 hour at 37° C. in a humidified 5% CO₂ atmosphere. Theadherent fraction was quantitated using MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays.In brief, after washing the wells, 10 μl of MTT (0.5 μg/ml finalconcentration) (Boehringer Mannheim Corp., Indianapolis, Ind.) was addedto each well and the plates were incubated at 37° C. for 4 hours toallow MTT to form formazan crystals by reacting with metabolicallyactive cells. The formazan crystals were solubilized overnight at 37° C.in a solution containing 10% SDS in 0.01 M HCl. The absorbance of eachwell was measured in a microplate reader (Labsystems) at 540 nm and areference wavelength of 690 nm. To translate the OD₅₄₀ values into thenumber of cells in each well, the OD₅₄₀ values were compared to those onstandard OD₅₄₀-versus-cell number curves generated for each cell line.The adherent fractions of cells treated with quinazoline derivativeswere compared to those of DMSO-treated control cells and the percentinhibition of adhesion was determined sing the formula:${\% \quad {Inhibition}} = {100*\frac{1 - \text{Adherent Fraction of Drug Treated Cells}}{\text{Adherent Fraction of Control Cells}}}$

Each treatment condition was evaluate d in duplicate in 3 independentexperiments. The IC50 values were calculated by non-linear regressionanalysis.

Results

As shown in FIG. 8, a significantly greater fraction of glioblastoma andmedulloblastoma cells adhered to plates precoated with laminin, type IVcollagen, or fibronectin than to uncoated or poly L-lysine-coatedcontrol plates. Of the four glioblastoma cell lines examined, U373 cellswere the most adhesive. Therefore, U373 cells were used in subsequentexperiments that were designed to examine the effects of variousquinazoline derivatives on integrin-mediated glioblastoma cell adhesion.

As shown in FIGS. 9A-9D, the novel quinazoline derivative4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P154)(but not the unsubstituted parent compound WHI-P258) inhibited theadhesion of U373 cells to laminin-, fibronectin-, and collagen-coatedplates in a dose-dependent fashion with mean IC50 values of 29.8±3.1 μM(N=3) for adhesion to fibronectin-coated plates, 36.1±3.5 μM (N=3) foradhesion to laminin-coated plates, and 42.7±2.5 μM (N=3) for adhesion tocollagen-coated plates. The 3′-bromo substitution on the phenyl ringlikely contributes to the activity of WHI-P154 since4-(4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P131] lackingthis bromo substituent was less potent than WHI-P154 (all IC50values: >50 μM). The 4′-hydroxyl substituent on the phenyl ring alsocontributed to the inhibitory activity of WHI-P154 since4-(3′-Bromophenyl)-amino-6,7-dimethoxyquinazoline[WHI-P79] which differsfrom WHI-P154 only by the lack of the 4′-hydroxyl group on the phenylring, was less potent (all IC50 values: >50 μM). Introduction of asecond bromo group at the 5′ position of the phenyl ring did not resultin improved inhibitory activity 4-(3′,5′-Dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline[WHI-P97]was not more potent than WHI-P154.

Example 7 Effect of WHI-P154 on EGF-induced Cell Adhesion

In addition to binding of cell surface receptors to ECM proteins andintegrin clustering, formation of focal adhesion plaques is alsoregulated by the activation of focal adhesion kinase by certain growthfactors upon binding to their receptors (Hatai, M., Hashi, H., Mogi, A.,Soga, H., Yokota, J., Yaoi, Y., 1994, FEBS Lett., 350:113-116; Ouwens,D. M., Mikkers, H. M., van der Zon, G. C., Stein Gerlach, M., Ullrich,A., Maassen, J. A., 1996, Biochem J., 318:609-614; Schaller, M. D.,Parsons, J. T., Curr. Opin. Cell; Zachary, I., 1997, Int. J. Biochem.,Cell Biol., 29:929-934). EGF is a potent mitogen for various brain tumorcells that express EGF receptor and are also shown to modulate theexpression of cell surface

To study the effects of quinazoline derivatives on EGF-stimulated celladhesion, the trypsinized and recovered cells were incubated withvarying concentrations ranging from 1 μM to 50 μM of quinazolines for 4hours at 37° C., then stimulated with 250 ng/ml of EGF and examined fortheir ability to adhere to poly-L-lysine coated plates.

For the EGF stimulation experiments, the cells were plated in thepresence of 250 ng/ml of EGF and allowed to adhere for 1 hour. Thenon-adherent cells were removed by gently washing the cells with PBS andthen the adherent fraction was quantitated as described above forExample 6.

Stimulation of U373 cells with EGF significantly increased the fractioncapable of adhering to poly-L-lysine coated plates from 33.2±5.2% to58.48±4.7% (P<0.02). A four-hour pretreatment of U373 cells withWHI-P154 not only completely prevented the EGF-induced adhesion but litalso reduced the adhesive fraction of U373 cells in a dose-dependentfashion far below the baseline levels despite the presence of EGF (FIG.10). Similar, albeit less potent, inhibitory effects were observed whencells were pretreated with WHI-P131.

Example 8 Substituted Quinazolines Inhibit Glioblastoma Cell Invasion

Glioblastoma Cell Invasion Through Matrigel Matrix

The in vitro invasiveness of glioblastoma cells was assayed using apreviously published method which employs Matrigel-coated Costar 24-welltranswell cell culture chambers (“Boyden chambers”) with 8.0-μm-porepolycarbonate filter inserts (Albini, A., Iwamoto, Y., Kleinman, H. K.,Martin, G. R., Aaronson, S. A., Kozlowski, J. M., and McEwan, R. N.,1987, Cancer Res., 47:3239-3245). The chamber filters were coated with50 μg/ml of Matrigel matrix, incubated overnight at room temperatureunder a laminar flow hood and stored at 4° C. Matrigel matrix is made upof several components of the extralcellular matrix (ECM), includingcollagens, laminin and proteo-glycans

On the day of the experiment, the coated inserts were rehydrated with0.5 ml serum-free DMEM containing 0.1% bovine serum albumin for 1-2hours. To study the effects of quinazoline derivatives on invasivenessof glioblastoma cells, exponentially growing cells were incubatedovernight with WHI-P97, WHI-P131 and WHI-P154 at various concentrationsranging from 1 μM to 50 μM. The cells were trypsinized, washed twicewith serum-free DMEM containing BSA, counted and resuspended at 1×10⁵cells/ml. 0.5 ml cell suspension containing 5×10⁴ cells in a serum-freeDMEM containing quinazoline compounds or vehicle was added to theMatrigel-coated and rehydrated filter inserts. Next, 750 μl of NIHfibroblast conditioned medium was placed as a chemoattractant in 24-wellplates and the inserts were placed in wells and incubated at 37° C. for48 hours. After the incubation period, the filter inserts were removed,the medium was decanted off and the cells on the top side of the filterthat did not migrate were scraped off with a cotton-tipped applicator.The invasive cells that migrated to the lower side of the filter werefixed, stained with Hema-3 solutions and counted under microscope. Fiveto 10 random fields per filter were counted to determine the mean (±SE)values for the invasive fraction. The invasive fractions of cellstreated with quinazoline derivatives were compared to those of DMSOtreated control cells and he percent inhibition of invasiveness wasdetermined using the formula:${\% \quad {Inhibition}} = {100\quad\left\lbrack \frac{1 - \text{Adherent Fraction of Drug Treated Cells}}{\text{Adherent Fraction of Control Cells}} \right\rbrack}$

Each treatment condition was evaluated in duplicate in 3 independentexperiments. IC50 values were calculated by non-linear regressionanalysis using Graphpad Prisin Software Version 2.0 (Graphpad SoftwareInc., San Diego, Calif.).

Results

As shown in FIG. 11, U373 glioblastoma cells were highly invasive inMatrigel-coated Boyden chambers. WHI-P154 inhibited the invasion of U373cells through the Matrigel matrix in dose-dependent fashion and it wasmore potent than WHI-P131 or WHI-P97 (FIG. 11). The mean IC50 valuesobtained from 3 independent experiments were 10.59±1.8 μM (range:9.57-11.64 μM) for WHI-P97, 7.07±1.8 μM (range 5.08-8.59 μM) forWHI-P131, and 4.46±0.8 μM (range: 3.53-5.01) for WHI-P154. The IC50values derived from the average values of three experiments were 9.58 μMfor WHI-P97, 7.95 μM for WHI-P131 and 5.2 μM for WHI-P154.

Example 9 Substituted Quinazolines Inhibit Focal Adhesion Plagues andActin Polymerization

Cytoskeletal organization and cellular adhesion are two crucialdeterminants of cell motility and these processes are controlled by thecomplex coordination of actin cytoskeletal rearrangement and changes infocal adhesions (Symons, M. H., and Mitchison, J. T., 1991, J. CellBiol., 114:503-513; Wang, Y. L., 1984, J. Cell Biol., 99:1478-1485;Bretcher, M. S., 1996, Cell, 87:601-606; Machesky, L. M., and Hall, A.,1997, J. Cell Biol., 138:913-926). Polymerization of actin filaments,formation of lamellepodia and filapodia at the leading edges areessential for the attachment and detachment of cells from the ECM andplay pivotal roles in cell motility and migration (Burridge, K., Fath,K., Kelly, G., and Turner, C., 1988, Ann. Rev. Cell Biol., 4:487-525;Burridge, K., Nuckolls, C., Otey, F., Pavalko, K., Simon, K., and TAurner, C., 1990, Cell Differ. Dev., 32:337-342). Formation of adhesionplaques is also important in this process because the polymerized actinfibers are tethered and linked to ECM at these junctions and the cellmovement is dependent on the strength of these focal adhesions. Moderatelevel of cellular adhesive strength is thought to be necessary for cellmigration (Burridge, K., Fath, K., Kelly, G., and Turner, C., 1988, Ann.Rev. Cell Biol., 4:487-525; Burridge, K., and Fath, K., 1989, Bioessays,10:104-108; Schwarzbauer, J. E., 1997, Curr. Biol., 7:292-294).Adhesions that are too strong may impair cell motility and adhesionsthat are too weak may not provide sufficient momentum to move the cellforward.

EGF-induced cell adhesion is brought about by enhanced formation ofFAK⁺/Actin⁺ focal adhesion plaques, which is triggered by redistributionof activated FAK. EGF-induced formation of focal adhesions inserum-starved U373 cells was examined by multicolor immunofluorescenceand confocal laser scanning microscopy using a murine monoclonalanti-FAK antibody (green fluorescence) and rhodamine-labeled phalloidinwhich stains actin (red fluorescence).

To evaluate the actin polymerization process, cells plated onpoly-L-lysine-coated plates were first serum-starved to depolymerize theactin stress fibers. Subsequently, cells were stimulated with fetalbovine serum to induce de novo stress fiber formation.

Fluorescence Microscopy

Immunofluorescence was used to study the effects of quinazolinederivatives on the formation of focal adhesion plaques andpolymerization of actin. Cells (obtained and maintained as described forExample 6) were plated on poly-L-lysine-coated glass-bottom 35 mm Petridishes (Mattek Corp., Ashland, Mass.) or fibronectin-coated cover slipsand maintained in DMEM supplemented with 10% FBS for 24 hrs. The mediumwas removed and the cells were washed twice with serum-free DMEM andincubated in the same medium for 16 hours. Following this serumstarvation, cells were incubated with varying concentrations ofWHI-P131, WHI-P154 or vehicle (0.1% DMSO) for 4-16 hours at 37° C. andthen stimulated either with 250 ng/ml of human recombinant EGF or 10%FBS for 15, 30, 60, 120 or 180 minutes at 37° C. At the end of the EGFstimulation, cells were washed twice with PBS, fixed in 2%paraformaldehyde in PBS (pH 7.2), permeabilized and non-specific bindingsites were blocked with 1.5% BSA and 0.1% triton X-100 in PBS for 30minutes.

To detect the focal adhesion plaques and actin, cells were incubatedwith a mixture of a mouse monoclonal antibody directed against the focaladhesion kinase at a dilution of 1:100 and rhodamine-labeled phalloidinat a dilution of 1:1000 for 1 hour at room temperature. Cells werewashed with PBS and incubated with a FITC-conjugated anti-mouse IgG(Amersham Corp., Arlington Heights, Ill.) for 1 hour (final dilution:1:40). Cells were washed with PBS, counterstained with TOTO-3 (MolecularProbes, INC.) at a dilution of 1:1000 for 10 minutes at roomtemperature, washed again with PBS and the coverslips were counted withVectashield (Vector Labs, Burlingame, Calif.). Subsequently, cells wereviewed with a confocal laser scanning microscope (Bio-Rad MRC 1024)mounted in a Nikon Labhophot upright microscope. Digital images weresaved on a Jaz disk and processed with Adobe Photoshop software (Adobesystems, Mountain View, Calif.). Prism software. Each experiment wasrepeated three times.

Results

As shown in FIGS. 12A, 12A′, 12B, 12B′, 12C, 12C′, a two-hourstimulation of serum-starved U373 cells with EGF resulted in asignificant decrease of the diffuse perinuclear/cytoplasmic FAK stainingaccompanied by the emergence of focal adhesion plaques with highintensity FAK staining (bright green fluorescence). These FAK⁺ adhesionplaques showed a strong phalloidin staining (bright red fluorescence)confirming the colocalization of actin. Notably, preincubation of U373cells with WHI-P154 (FIG. 12B) or WHI-P131 (data not shown) at a 10 μMconcentration prevented the formation of FAK⁺/Actin⁺ focal adhesionplaques after EGF stimulation.

To evaluate the actin polymerization process, cells plated onpoly-L-lysine-coated plates were first serum-starved to depolymerize theactin stress fibers. Subsequently, cells were stimulated with fetalbovine serum to induce de novo stress fiber formation. As shown in FIGS.13A-C, a two-hour stimulation of serum-starved U373 cells with fetalbovine serum (10% v/v) resulted in a marked increase in polymerizedactin stress fibers. Pretreatment of serum-starved U373 cells withWHI-P154 inhibited serum-induced actin polymerization (FIG. 13C).Similar results were obtained with WHI-P131 but not with theunsubstituted dimethoxy quinazoline compound WHI-P258 (data not shown).

In summary, the data provided in Examples 6-9 demonstrate theeffectiveness of substituted quinazolines in the inhibition ofglioblastoma cell adhesion and migration, key factors for tumor cellmetastasis. The most potent inhibitory agents were WHI-P154 andWHI-P131. Both compounds inhibited adhesion and migration at micromolarconcentration.

A complex network of intracellular molecules including receptor tyrosinekinases and Src family tyrosine kinases in cooperation with severalextracellular factors such as substratum to which cell adhere andexternal factors, regulates the cell adhesion and motility (Finchman V.J., and Frame, M. C., 1998, EMBO J., 17:81-92). Activation of integrinfamily adhesion receptors upon binding to specific extracellular matrixproteins has been shown to enhance the phosphorylation of integrins andactivation of several intracellular signaling proteins including mitogenactivated protein kinase, FAK, Src tyrosine kinases as well asp130^(cas), talin, paxillin, and cortactin which were identified assubstrates for the Src tyrosine kinase (Cobb, B. S., Schaller, M. D.,Leu, T. H., and Parsons, J. T., 1994, Mol. Cell Biol., 14:147-155; Chen,Q., Lin, T. H., Der, C. J., Juliano, R. L., 1996, J. Biol. Chem.,271:18122-18127; Klemke, R. L., Cai, S., Giannini, A. L., Gallagher, P.J., Lanerolle, P. D., and Cheresh, D. A., 1997, J. Cell Biol.,137:481-492; Petch, L. A., Bockholt, S. M., Bouton, A., Parsons, J. T.,Burridge, K., 1995. J. Cell Sci., 108:1371-13719; Chrzanowska-Wodnicka,M., and Burridge, K., 1996. J. Cell Biol., 133(6):1403-15; Miyamoto, S.,Akiyama, S. K., Yamada, K. M., 1995, Science, 267:883-5; Miyamoto, S.,Teramoto, H., Gutkind, J. S., and Yamada, K. M., 1996, J. Cell Biol.,135:1633-1642; Chen, H. C., Appeddu, P. A., Parsons, J. T., Hildebrand,J. D., Schaller, M. D., and Guan, J. L., 1995, J. Biol. Chem.I,16995-16999). Subsequently, the adhesion of cell is strengthened byredistribution of activated Src kinase and focal adhesion kinase tofocal adhesions, recruitment and aggregation of activated intracellularproteins such as paxillin, talin, viniculin and clustering of integrins(Burridge, K., Fath, K., Kelly, G., and Turner, C., 1988, Ann. Rev. CellBiol., 4:487-525; Burridge, K., and Fath, K., 1989, Bioessays,10:104-108; Finchman, V. J., and Frame, M. C., 1998, EMBO J., 17:81-92).FAK is also activated by the binding of certain growth factors to theirreceptors in a mechanism that is independent of integrin activation(Hatai, M., Hashi, H., Mogi, A., Soga, H., Yokota, J., Yaoi, Y., 1994,FEBS Lett., 350:113-116; Ouwens, D. M., Mikkers, H. M., van der Zon, G.C., Stein Gerlach, M., Ullrich, A., Maassen, J. A., 1996, Biochem J.,318:609-614).

In experiments not shown here, WHI-P154 was found to be a potentinhibitor of the EGF-R tyrosine kinase as well as Src family tyrosinekinases (Liu and Uckun, manuscript in preparation). Therefore, it wasinitially postulated that the effects of WHI-P154 on glioblastoma cellswas due to its tyrosine kinase inhibitory properties. Surprisingly,however, WHI-P79 and WHI-P131, which are equally potent inhibitors ofthe EGF-R and Src family tyrosine kinases were not as effective asWHI-P154 and the broad spectrum tyrosine kinase inhibitor genistein didnot affect glioblastoma cell adhesion and motility at concentrationswhich abrogate the enzymatic activity of the EGF-R kinase and Src familytyrosine kinases. Thus, the effects of WHI-P154 on U373 cells cannot beexplained by its tyrosine kinase inhibitory properties alone.

Example 10 WHI-P154 Inhibits Glioblastoma Cell Migration from Spheroids

U373 glioblastoma spheroids of 200 to 400 micrometers in diameter weretreated with WHI-P154 in 0.1% DMSO at varied concentrations. Cells wereincubated with the inhibitor or with control DMSO in the absence ofinhibitor compound for two hours, and then transferred tofibronectin-coated coverslips. The spheroids were then incubated in DMEMcontaining WHI-P154 at 37° C. for 48 hours.

As shown in FIGS. 14A-D, treatment of glioblastoma spheroids withWHI-P154 significantly inhibited cell migration from the spheroid ascompared with the untreated control and in a dose-dependent manner. TheFigure shows the following dosage treatments: A: control; B: 2.5 μM; C:4.5 μM; and D: 10 μM.

Example 11 Cytotoxic Activity of WHI-P292

The novel compound WHI-P292 was assayed for cytotoxic activity in theMTT cell survival assay, as described above for Example 3. As shown inFIG. 15, this compound demonstrated potent cytotoxic activity againstglioblastoma cells, with an IC₅₀ of 38.22 μM.

All publications, patents, and patent documents described herein areincorporated by reference as if fully set forth. The invention describedherein may be modified to include alternative embodiments. All suchobvious alternatives are within the spirit and scope of the invention,as claimed below.

We claim:
 1. A method comprising contacting brain tumor cells with abrain tumor cell apoptosis inducing amount of a compound of the formula:


2. A method comprising contacting brain tumor cells with a brain tumorcell metastases preventing amount of a compound of the formula: